JP2011518199A - Human umbilical cord perivascular cells genetically modified for prevention or treatment of biological or chemical agents - Google Patents

Abstract

  The present invention prevents or prevents diseases or disorders caused by biological or chemical agents in a subject (eg, a mammal such as a human) by administering genetically modified human umbilical perivascular cells. Provide a method of treatment.

Description

  The present invention prevents or treats a disease or disorder caused by a biological or chemical agent in a subject (eg, a mammal such as a human) by administering genetically modified human umbilical cord perivascular cells. Provide a method. Also provided are genetically modified human umbilical cord perivascular cells useful in such methods and pharmaceutical compositions comprising them.

  The production of recombinant proteins for therapeutic proteins with clinical value has been a great technical and commercial success of the biotechnology industry. Monoclonal antibodies (MAbs) are the fastest growing pharmaceutical sector with worldwide sales of $ 20.6 billion in 2006. The top six selling US monoclonal antibodies are Rituxan® alone, with over $ 1 billion in sales per year for one year and the top selling $ 4.7 billion.

  The use of monoclonal antibodies for national defense is of great interest to institutions ordered to treat disasters caused by biological weapons (and the massive spread of infectious diseases such as SARS). Military and civilian research programs have identified a number of therapeutic monoclonal antibodies, and in some cases, the process of developing them as biomedical medical countermeasures has begun.

  When deploying monoclonal antibodies as a top priority, two issues must be addressed: high drug costs and cumbersome delivery systems. The cost of producing a “biologic” is high. Recombinant proteins such as monoclonal antibodies are produced by costly processes including large-scale cGMP production and eukaryotic / prokaryotic fermentation systems, followed by downstream purification and refining of the final bulk pharmacologically active bulk powder. The Second, the delivery of monoclonal antibodies and other protein-based drugs by injection is a therapeutic barrier. The large-scale use of monoclonal antibodies by conventional injection or injection is impractical within the time and personnel constraints of battlefield bioweapon attacks or terrorist scenarios.

  The present invention provides biology by administering genetically modified HUCPVCs such that the expression of oligonucleotides or polypeptides in HUCPVCs is increased relative to unmodified human umbilical cord perivascular cells (HUCPVCs). A method is provided for protecting a subject against attacks from chemical or chemical agents. In another aspect of the invention, genetically modified HUCPVCs useful in such methods and pharmaceutical compositions comprising such genetically modified cells are also provided. The subject may be a vertebrate such as a mammal (eg, a human). HUCPVC may be allogeneic or xenogeneic for the subject to which it is administered.

  In one embodiment of the invention, genetically modified HUCPVC is administered to a subject to protect against attack from biological agents. The biological agent may be a pathogen such as a bacterium, virus, fungus, or parasite. The bacteria are Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli, Klebsiella pneumoniae, Brucella bursell, ), Or Bacillus anthracis. The viruses are Gadgets Gully virus, Kadam virus, Kazanur forest disease virus, Langat virus, Omsk hemorrhagic fever virus, Poissan virus, Royal farm virus, tick-borne encephalitis virus, leap Disease virus, Meban virus, Saumarez Reef virus, Tyureny virus, Aroa virus, Dengue virus, Kedougo virus, Kashipa core virus C virus), Koutango virus, Japanese encephalitis virus, Murray Valley Flame virus, St. Louis encephalitis virus, Ustu virus, West Nile virus, Yaonde virus, Cocobella virus, Bugaza virus, Ileus virus, Israel Turkey meningoencephalomyelitis virus, Untaya virus, Tenbus virus, Zika virus, Banji virus, Bowfish Bovine virus, Edgehill virus, Jugra virus, Savoia virus, Sepic virus, Uganda S virus, Weselsbron virus, Yellow fever virus, Entebe bat virus, Yokose virus, Apoi virus, Cowbone ridge virus (Cowbone) Ridge virus), ftiapa virus, modok virus, salvia virus (Sal Vieja virus), Saint-Pelita virus , Bucaras bat virus, Curry Island virus, Dakar bat virus, Montanamyotis leukoencephalitis virus, Phnom Penh bat virus, Rio Bravo virus, Venezuelan equine encephalitis virus (VEE), Eastern equine encephalitis virus (EEE), Western equine encephalitis virus (WEE), Ebola virus, Marburg virus, smallpox virus, vaccinia virus, Lassa virus, Ipy virus, lymphocytic choriomeningitis virus (LCMV), mobara virus (Mobala virus), mopeia virus , Amapari virus, Flexal virus, Guanarito virus, Junin virus, latinovirus, Machupo virus, Oliveros virus (Olivero virus) s virus), Paranavirus, Pickinde virus, Pirital virus, Sabia virus, Tacaribe virus, Tamiamivirus, and Whitewater Arroyovirus, Sinnombre virus, HanTurn virus, Rift Valley fever virus, Crimea Congo hemorrhagic fever virus, Dugbe virus, herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), Kaposi sarcoma-associated herpes virus (KSHV), influenza virus A, H5N1 bird Influenza virus, influenza virus B, influenza virus C, severe acute respiratory syndrome (SARS) virus, rabies virus, or vesicular stomatitis virus It may be a VSV). Fungi are Aspergillus, Blastomyces dermatitis, Candida, Coccidioides immitis, Cryptococcus neoformans, Cryptococcus neoformans var. capsulatum), Paracoccidioides brasiliensis, Sporotix schenckii, Zygomycetes spp., Absidia It may be a Corbifera (Absidia corymbifera), Rhizomucor pusillus, or Rhizopus arhizus. Parasites are Toxoplasma gondii, Plasmodium falciparum, Plasmodium falciparum (P. vivax), Plasmodium falciparum (P. ovale), Plasmodium falciparum (P. malaria) ), Trypanosoma spp., Or Legionella spp.

  In another embodiment of the invention, genetically modified HUCPVC is administered to a subject to protect against attack from chemical agents. Chemical agents include ricin, diphtheria toxin, E. coli enterotoxin, Vibrio cholerae enterotoxin, Staphylococcal enterotoxin, Streptococcal enterotoxin, or botulinum toxin, etc. May be a bacterial, viral, fungal, or parasitic toxin. The chemical agent may be synthetic or naturally derived. The chemical agent may be a nerve agent, a blister agent, a blood agent, or a respiratory agent. The chemical agent may be nitrogen mustard, sulfur mustard, acetylcholinesterase inhibitor, or lewisite. Chemical agents include saxitoxin, bis (2-chloroethyl) ethylamine, 2-chlorovinyldichloroarsine, 2-chloroethylchloromethyl sulfide, bis (2-chloroethyl) sulfide, sarin (GB), cyclosaline (GF), soman. (GD), tabun (GA), VX, amiton, PFIB, 3-quinuclidinyl benzylate, phosgene, diphosgene, cyanogen chloride, hydrogen cyanide, or chloropicrin.

  In other embodiments of the present invention, HUCPVC is genetically modified to produce antibodies, antibody fragments, microbial antigens, cytokines or growth factors, hormones, clotting factors, drug-resistant or antiviral resistant polypeptides, antitoxin drugs, antioxidants, Polypeptides such as receptors or ligands, immunomodulators, detectable labels, or cellular factors are expressed. The polypeptide can be endogenous or non-endogenous to HUCPVC. In still other embodiments, HUCPVC can be genetically modified to express more than one polypeptide. In yet other embodiments, HUCPVC can synthesize and secrete antibodies or antibody fragments, which can be recombinant, humanized, or monoclonal. The antibody or antibody fragment may be a single chain antibody (scFv), Fab, Fab'2, scFv, SMIP, diabody, nanobody, aptamer, or domain antibody. In still other embodiments, the cytokine or growth factor is tumor necrosis factor α (TNF-α), TNF-β, interferon-α (IFN-α), IFN-β, IFN-γ, interleukin 1 (IL- 1), IL-1β, interleukin 2-14, granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), RANTES, MIP-1α), transforming growth factor-β ( TGF-β), platelet derived growth factor (PGDF), insulin-like growth factor (IGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), erythropoietin (EPO), or thrombopoietin (TPO). Hormones are angiotensinogen, angiotensin, parathyroid hormone (PTH), basic fibroblast growth factor-2, luteinizing hormone, follicle stimulating hormone, adrenocorticotropic hormone (ACTH), vasopressin, oxytocin, somatostatin, gastrin Or cholecystokinin, leptin, atrial natriuretic peptide, epinephrine, norephinephrine, dopamine, calcitonin, or insulin. The clotting factor can be Factor VII, Factor VIII, Factor IX, or fibrinogen. Enzymes include butyrylcholinesterase (BChE), adenosine deaminase, glucocerebrosidase, α-1 antitrypsin, viral thymidine kinase, hypoxanthine phosphoribosyltransferase, manganese superoxide dismutase (Mn-SOD), catalase, copper-zinc-super It may be oxide dismutase (CuZn-SOD), extracellular superoxide dismutase (EC-SOD), glutathione reductase, phenylalanine hydroxylase, nitric oxide synthase, or paraoxynase. The receptor or ligand may be a T cell receptor (TCR), LDL receptor, surface-bound immunoglobulin, soluble CD4, cystic fibrosis transmembrane conductance receptor (CFTR), or Fc receptor. Immunomodulators include CTLA-4, VCP, PLIF, LSF-1, Nip, CD200, uromodulin, CD40L (CD154), FasL, CD27L, CD30L, 4-1BBL, CD28, CD25, B7.1, B7.2, Or it may be OX40L. The detectable label may be green fluorescent protein (GFP). The cellular factor may be cytochrome b, ApoE, ApoC, ApoAI, MDR, tissue plasminogen activator (tPA), urokinase, hirudin, β-globulin, α-globulin, HbA, ras, src, or bcl. A polypeptide may be a cellular protein that acts as an antigen, thereby generating an immune response against a biological or chemical agent in a subject.

  In yet another embodiment of the invention, HUCPVC is genetically modified to express an oligonucleotide, eg, an RNA interference (RNAi) molecule that can inhibit viral replication or infection. The RNAi molecule may be a small inhibitory RNA (siRNA) or a short hairpin RNA (shRNA) molecule. Oligonucleotides can be endogenous or non-endogenous to HUCPVC. In other embodiments, HUCPVC can be genetically modified to express more than one oligonucleotide.

In another embodiment of the invention, the subject has been exposed to a biological or chemical agent prior to administration of the genetically modified HUCPVC of the invention. A subject can be administered a single dose of HUCPVC or multiple doses of HUCPVC. HUCPVC can be administered as a vaccine to protect subjects in need thereof. HUCPVC is intravenous, intramuscular, oral, inhalation, parenteral, intraperitoneal, intraarterial, transdermal, sublingual, nasal, buccal, liposome, fat, ocular, intraocular, subcutaneous, subarachnoid, It can be administered locally or locally. 42. Subjects can be administered 10 < ¹ > to 10 < ¹³ > HUCPVVC per dose, or 10 < ³ > to 10 < ⁸ > HUCPVVC per dose.

  In other embodiments of the invention, the genetically modified HUCPVC administered to the subject persists for more than 1 week, 1 month, or 2 months. HUCPVC can avoid immune recognition in a subject.

  In yet another embodiment of the invention, the genetically modified HUCPVC is administered in combination with at least one mesenchymal stem cell (MSC) that is not HUCPVC. MSCs can be genetically modified to increase the expression of oligonucleotides or polypeptides in MSCs compared to non-genetically modified MSCs. MSCs can be isolated from bone marrow, umbilical cord blood, embryonic yolk sac, placenta, skin, or blood. MSCs can be genetically modified to express oligonucleotides or polypeptides that are endogenous or non-endogenous to HUCPVC.

  In another embodiment of the invention, the genetically modified HUCPVC can be administered to a subject in combination with one or more therapeutic agents that enhance or prolong the prophylactic or therapeutic effect of HUCPVVC treatment. The therapeutic agent may be, for example, a cytokine, an antiviral agent, an immunostimulant, or an immunosuppressant.

  In yet another embodiment of the invention, the genetically modified HUCPVC is administered with a pharmaceutically acceptable carrier or excipient.

  In another embodiment of the invention, the genetically modified HUCPVC is provided in a kit for administration to a subject in need of treatment or protection from a biological or chemical agent.

  In general, the present invention provides the use of genetically modified HUCPVCs for the preparation of a medicament for the prevention or treatment of a disease or disorder caused by a biological or chemical agent in a subject.

Definitions The term “antibody” used interchangeably herein includes whole antibodies or immunoglobulins and any antigen binding fragment or single chain thereof. As used herein, an antibody can be mammalian (eg, human or mouse), humanized, chimeric, recombinant, and can be produced synthetically or isolated naturally. The antibodies of the present invention include all known types of antibodies and other protein backbones with antibody-like properties. For example, the antibody may be a human antibody, a humanized antibody, a bispecific antibody, a chimeric antibody, or a protein backbone with antibody-like properties such as fibronectin or ankyrin repeat. The antibody may be a Fab, Fab′2, scFv, SMIP, diabody, nanobody, aptamer, or domain antibody. The antibody can have any of the following isotypes: IgG (eg, IgG1, IgG2, IgG3, and IgG4), IgM, IgA (eg, IgA1, IgA2, and IgAsec), IgD, or IgE.

The term “antibody fragment” as used herein refers to a fragment of one or more antibodies that retain the ability to specifically bind to an antigen. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a monovalent fragment consisting of a Fab fragment, V _L , V _H , C _L , and C _H 1 domains, (ii) F (Ab ′) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region, (iii) an Fd fragment consisting of V _H and C _H 1 domains, (iv) V of the single chain arm of the antibody Fv fragment consisting of _L and V _H domains, (v) dAb containing V _H and _VL domains, (vi) dAb fragment (Ward et al., Nature 341: 544-546 (1989)), which consists of V _H domains, (Vii) a dAb consisting of a _VH or _VL domain, (viii) an isolated complementarity determining region (CDR), and (ix) optionally linked by a synthetic linker. There are, but are not limited to, combinations of two or more isolated CDRs that are capable. In addition, the two domains of the Fv fragment, _VL and _VH, are encoded by separate genes, which use recombinant methods in which the _VL and _VH regions are paired and monovalent. They can be linked by a synthetic linker that is capable of generating them as a single protein chain that forms a molecule (known as single chain Fv (scFv); for example, Bird et al., Science 242: 423). 426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)).

  The term “effective amount” or “effective amount” or “therapeutically effective amount” means the desired result, eg, prevention or treatment of bacterial or viral infection, reduction of blood coagulation, or the action of a poison (eg, snake venom) By an amount of genetically modified HUCPVC sufficient to cause attenuation.

  “Gene modified HUCPVC” means when at least one polypeptide (eg, antibody, cytokine, or hormone) or oligonucleotide (eg, siRNA) is recombinantly expressed and administered to a human (eg, soldier) By human umbilical cord perivascular cells capable of preventing or treating diseases or disorders caused by pathogenic microorganisms or chemical agents (eg, toxins). This polypeptide or oligonucleotide is recombinantly produced by HUCPVC after introduction (eg, transfection or transduction) of the gene sequence of the polypeptide or oligonucleotide into HUCPVC.

  As used herein, the term “human antibody” refers to, for example, Kabat et al. (“Sequences of proteins of Immunological Interest”, 5th edition, US Department of Health and Human. Services, NIH Publication No. 91- 3242 (1991)), an antibody, or fragment thereof, wherein both the framework and CDR regions have variable regions derived from human germline immunoglobulin sequences. Shall be included. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. Human antibodies may contain amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, mutations introduced in vitro by random or site-specific mutations or by somatic mutations in vivo). There is. However, as used herein, the term “human antibody” refers to an antibody in which a CDR sequence derived from the germline of another mammalian species such as a mouse is grafted onto a human framework sequence (ie, a humanized antibody or antibody). (Fragment) is not included.

  The term “humanized antibody” comprises a variable framework region substantially derived from a human immunoglobulin or antibody and a complementarity determining region substantially derived from a non-human immunoglobulin or antibody (eg, at least one CDR). Refers to any antibody or antibody fragment comprising at least one immunoglobulin domain having a variable region.

  "Interferon" refers to a mammalian (eg, human) interferon-α, -β, -γ, or -tau polypeptide, or a biologically active fragment thereof, such as IFN-α (eg, IFN-α-1a; US Patent Application No. 2007/0274950, incorporated herein by reference), IFN-α-1b, IFN-α-2a (PCT Application No. WO07 / 044083, incorporated herein by reference). Reference), and IFN-α-2b), IFN-β (eg, as described in US Pat. No. 7,238,344, incorporated herein by reference; US patents incorporated herein by reference). IFN-b-1a (AVONEX® and REBIF®) described in US Pat. No. 6,962,978, and IFN-β-1 (BETASERON®, U.S. Pat. No. 4,588,585, U.S. Pat. No. 4,959,314, U.S. Pat. No. 4,737,462, and U.S. Pat. No. 4,450,103), IFN-g, and IFN-t (US Pat. No. 5,738,845 and US Patent Application Publication No. 2004/0247565, which are incorporated herein by reference). And 2007/0243163).

  “Pharmaceutically acceptable carrier” means a carrier that is physiologically acceptable to the subject (eg, human) to be treated while retaining the therapeutic properties of the genetically modified HUCPVC administered therewith. One exemplary pharmaceutically acceptable carrier is saline. Other physiologically acceptable carriers and their formulations are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences, (18th edition), A.S., incorporated herein by reference. Edited in Gennaro, 1990, Mack Publishing Company, Easton, PA.

  “Treatment” refers to the progression or severity of a disease or disorder (eg, an infectious disease caused by a pathogenic microorganism or toxin), or the progression, severity, or severity of one or more symptoms of a disease or disorder in a subject (eg, a human), or Decrease in frequency (eg, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 99 %, Or even 100%).

  The present invention relates to genetically modified human umbilical cord perivascular cells (HUCPVCs), medically useful compositions containing them, and biological agents (eg, Inhibiting, reducing, preventing, or treating attacks by pathogens such as bacteria, viruses, fungi, and parasites) or chemical agents (eg, biological or chemical (eg, synthetic) toxins) Of their administration. In addition, genetically modified HUCPVCs may be exposed to or exposed to pathogens or biological or chemical toxins, respectively, including toxins used in biological or chemical weapons. Can be administered prophylactically or therapeutically. The method of the present invention is useful for the prevention or treatment of patients, eg, military personnel (eg, soldiers), medical personnel (eg, doctors or nurses), or civilians who are at high risk of exposure to biological or chemical weapons. Especially suitable. Furthermore, the present invention relates to military personnel or medical personnel who reside in regions of the world where prevalent infectious agents (eg, malaria, flaviviruses such as dengue virus, West Nile virus, and yellow fever virus) present significant health risks. Prophylaxis or treatment of the person.

  Such as antibodies, antibody fragments, cytokines, hormones, coagulation factors, immunomodulators, detectable labels, or enzymes that genetically modify HUCPVC to provide a prophylactic or therapeutic benefit to the subject being treated (eg, human) Polypeptides (eg, human polypeptides) can be expressed. In addition, HUCPVC can be genetically modified to express oligonucleotides (eg, RNAi molecules) that modulate (eg, inhibit) cellular processes of the subject to be treated or pathogenic microorganisms or toxins present therein. . HUCPVC can be genetically modified to express one or more therapeutic polypeptides or oligonucleotides for the prevention or treatment of one or more diseases or disorders resulting from exposure to pathogenic microorganisms or toxins It is.

  Co-administering genetically modified HUCPVC with one or more diagnostic or therapeutic agents (eg, immunomodulators such as interferon-α) to enhance or prolong the prophylactic or therapeutic properties of HUCPVVC treatment Can do. HUCPVC can also be administered in combination with one or more pharmaceutically acceptable carriers or excipients and can be formulated intravenously, intramuscularly, orally, inhaled, parenterally, intraperitoneally, intraarterially, transdermally. Skin, sublingual, nasal, buccal, liposome, fat, ocular, intraocular, subcutaneous, subarachnoid, topical, or local administration. In a further aspect, the present invention provides kits with instructions for the prophylactic or therapeutic treatment of mammals with one or more genetically modified HUCPVC populations.

Human umbilical cord perivascular cells (HUCPVC)
Human umbilical cord perivascular cells (HUCPVCs) are non-hematopoietic, mesenchymal populations of multipotent cells obtained from surrounding regions within the walton jelly of human umbilical cords (see, eg, Sarugaser et al. ) Cells: Source of mesenchymal progenitor cells (see Human source cord perivascular (HUCPV) cells): A source of mesenchymal progenitors ", Stem Cells 23: 220-229 (2005)). US Patent Application Publication No. 2005/0148074 and International Patent Application Publication No. WO 2007/128115 describe methods for isolation and in vitro culture of HUCPVC, which are incorporated herein by reference. HUCPVC is further characterized by a relatively rapid growth that exhibits a doubling time (serum dependent) of about 20 hours in each of the second to seventh generations when cultured under standard adhesion conditions. Phenotypically, HUCPVC is expressed at Oct4-, CD14-, CD19-, CD34-, CD44 +, CD45-, CD49e +, CD90 +, CD105 (SH2) +, CD73 (SH3) +, CD79b-, HLA-G at the time of harvest. Characterized as-, CXCR4 +, and c-kit +. Furthermore, HUCPVC is positive for CK8, CK18, CK19, PD-L2, CD146 and 3G5 (ambient cell markers) at high levels compared to cell populations extracted from sources of Walton Jerry other than the surrounding area.

Advantages of HUCPVC When used to recombinantly express a polypeptide or oligonucleotide (eg, a human polypeptide or oligonucleotide), genetically modified HUCPVC offers several advantages over other cell-based therapies . Since HUCPVCs are less immunogenic when administered to allogeneic or xenogeneic hosts, they have an increased life span within the host compared to other allogeneic or xenogeneic cells. HUCPVC also has a demonstrated gene expression modality that results in a therapeutically significant level of the protein or oligonucleotide of interest (eg, a recombinant polypeptide or oligonucleotide in which HUCPVC is genetically modified and expressed). Furthermore, although HUCPVCs grow rapidly, they have a lower risk of proliferative disorders compared to other cell-based gene therapy vehicles. Each of these advantageous properties of genetically modified HUCPVC for preventing or treating a subject (eg, a human) is discussed in detail below.

  The low immunogenicity of genetically modified HUCPVCs makes them ideal vehicles for administration to vertebrate subjects, eg mammals such as humans, and in particular allogeneic or xenogeneic recipients. HUCPVCs have been shown to have low immunogenicity based on their ability to avoid detection by the host immune system (see, eg, Sarugaser et al. (2005) and US Patent Application Publication No. 2005/0148074). ). Thus, for example, HUCPVCs collected from humans (ie, donors) can be cultured in vitro, without inducing a homospecific immune response against the genetically modified HUCPVCs in the host, Can be administered to HLA-mismatched humans (ie, hosts) (eg, Ennis et al., “In vitro immunological properties of human cord perivascular cells 10”) (2) See 174-181 (2008)). Thus, the genetically modified HUCPVC can be administered to a heterologous human population or even to a heterogeneous population without loss of therapeutic efficacy due to activation of the host immune system. Furthermore, the ability to use HUCPVC in virtually any vertebrate (eg, a mammal such as a human) has made extensive preparation and storage for use in an emergency situation (eg, an epidemic of infectious disease) (ie, "Stockpile").

  The low immunogenicity of HUCPVC results in an increased life span of these cells in vivo in the treated host compared to other allogeneic or xenogeneic cells. It has been documented that similar mesenchymal cells persist in human hosts for several years when delivered allogeneically (Le Blanc et al., “After Intrauterine Transplantation in Patients with Severe Osteogenesis Imperfection. Fetal mesenchymal stem-cell engraftment in bone after in vitro transplantation in a patient with 7 7 (from 7 to 79) HUCPVC can be used at least several weeks to several months after injection (eg, 2 weeks, 4 weeks, 6 weeks, 2 months or more), vertebrates (eg, mammals such as humans). ) Can be expected to persist in the host. The lifetime of HUCPVCs used to provide therapeutic or prophylactic polypeptides or oligonucleotides (eg, by providing viral polypeptides or oligonucleotides) provides advantages over other vaccination or therapeutic techniques. While conventional vaccines or therapeutic agents require multiple administrations to provide a protective or therapeutic effect in an individual, a therapeutically effective amount of genetically modified HUCPVC can be administered to an individual in a single dose. Alternatively, two or more doses of genetically modified HUCPVC can be administered to provide prevention or treatment. The lifetime of genetically modified HUCPVCs used to prevent or treat diseases or disorders caused by pathogenic microorganisms or toxins allows them to receive multiple doses of therapeutic agents or vaccines due to the uncertain nature of military deployment It is particularly useful for administration to military personnel (eg, soldiers) who are unable to do so.

  Another advantageous property of HUCPVCs is that they can be easily genetically modified by several standard transfection and transduction techniques to allow recombinant expression of therapeutic polypeptides or oligonucleotides. is there. As described further herein, gene transfer uses viral vectors (eg, adenoviruses and lentiviruses) and nucleic acid transfections (eg, DNA plasmids combined with liposomes, cationic media, or electroporation). Can be implemented.

  Unlike many other mesenchymal stem cell populations that typically require bone marrow donation, HUCPVC can be reliably recovered from human umbilical cords that are normally discarded after birth. In industrialized countries, human umbilical cord blood products are now routinely collected and stored for possible future autologous or allogeneic transplants. Thus, the recovery of HUCPVC for growth and genetic modification by the method of the present invention is free of many theoretical constraints on the recovery of other mesenchymal stem cell populations.

  Finally, HUCPVC has a short population doubling time that allows rapid and large-scale preparation of genetically modified HUCPVCs for administration to mammals (eg, humans) in need thereof (eg, Sarugaser Et al., 2005). HUCPVC is substantially devoid of the enzyme telomerase and therefore has a minimal risk of developing a proliferative disease. This is because these cells cannot divide beyond a predetermined number of divisions before apoptosis occurs. In animal studies, HUCPVC is not known to produce tumors even when administered in orders of magnitude greater than clinically accepted.

Expression of Recombinant Polypeptides and Oligonucleotides In the present invention, when HUCPVC is genetically modified and given in a therapeutically effective amount, the genetically modified HUCPVC acts as a “stealth cell” and is biological (eg, pathogenic One or more polypeptides (eg antibodies, cytokines or hormones) or oligonucleotides (eg siRNA molecules) so as to inhibit, reduce, prevent or treat attack by microorganisms) or chemical (eg toxins) agents ) Can be expressed. Immunomodulatory oligonucleotides or polypeptides can also be expressed in HUCPVC to modulate (eg, increase or decrease) the host immune response. Polypeptides expressed in HUCPVC can be secreted or presented on the plasma membrane surface (eg, membrane-bound receptors or ligands). One or more oligonucleotides or polypeptides can be co-expressed in one HUCPVC to allow treatment or protection of one or more biological or chemical agents.

Antibodies and Antibody Fragments The present invention further provides for the modification of antibodies (eg, humanized monoclonal antibodies) or antibody fragments by genetically modified HUCPVCs that specifically bind and neutralize pathogenic microorganisms or chemical agents (eg, toxins). Give expression. As shown in Example 1, vertebrates (eg, mammals) that have been or have been exposed to pathogenic microorganisms or chemical agents, respectively, using HUCPVCs that express antibodies or antibody fragments. , Humans (eg, soldiers), etc.) can be treated or protected. For example, exemplary antibodies that can be used to modulate the immune system include TNF inhibitors (eg, infliximab, adalimumab, certolizumab pegol), alemtuzumab, aferimomab, acelizumab, atrizumab, atrolimumab, basiliximab, belimumab , Bertilimumab, Cedelizumab, Clenolimimab, Garitanimab, Gantimumab, Gartinimab, Gantimumab, Gantimumab , Relderimumab, Lumilimimab, Masrimomab, Mepolizumab, Meterimumab Mororimumabu, muromonab -CD3, natalizumab, Nererimomabu, ocrelizumab, Ozurimomabu, omalizumab, Oterikishizumabu, Pasukorizumabu, pexelizumab, Ritsukisumabu, Resurizumabu, Roberizumabu, Rupurizumabu, siplizumab, Tarizumabu, Teri mode Mabu Ali Tox, Tenerikishimabu, Tepurizumabu, tocilizumab, Torarizumabu, Baparikishimabu, Beparimomabu , Bicilizumab, zanolimumab, diralimumab, and zolimoumab ritox.

Microbial antigens as vaccines HUCPVCs expressing one or more antigens from microbial pathogens (eg, bacteria, viruses, fungi, or parasites) can be used as vaccines to induce a protective or therapeutic immune response in the subject being treated. can do. Upon administration, the genetically modified HUCPVC expresses a microbial antigen that is recognized as foreign by the host immune system. Generation of primary immune responses against antigens, including activation of adaptive immune responses (eg, host antibodies and T cells) allows for the generation of strong and long-term secondary responses upon infection by microbial pathogens. The use of bacterial, viral, fungal, and parasitic polypeptides in vaccines and the identification of immunogenic antigens derived from these microorganisms that are suitable for expression in HUCPVC according to the methods of the invention are known in the art. ing.

Cytokines and Growth Factors The present invention provides for the expression of cytokines and growth factors by genetically modified HUCPVC. Many cytokines have immunomodulatory properties and can be used to enhance the immune response or reduce the immunopathological response. Examples of cytokines and growth factors that can be expressed in HUCPVC include tumor necrosis factor (TNF) such as TNF-α, interferons (eg, interferon-α, interferon-β, and interferon-γ), interferon Leukins (eg, IL-1, IL-1β, and interleukins 2-14), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), chemokines (eg, RANTES and MIP) -1α), a member of the transforming growth factor-β (TGF-β) superfamily, platelet derived growth factor (PGDF), insulin-like growth factor (IGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) , Keratinocyte growth factor (KGF), erythropoietin ( EPO), and thrombopoietin (TPO), but are not limited to these.

Hormones The present invention provides for the expression of hormones by genetically modified HUCPVC. Examples of hormones that can be expressed in HUCPVC include angiotensinogen, angiotensin, parathyroid hormone (PTH), basic fibroblast growth factor-2, luteinizing hormone, follicle stimulating hormone, adrenal cortex These include, but are not limited to, stimulating hormone (ACTH), vasopressin, oxytocin, somatostatin, gastrin, cholecystokinin, leptin, atrial natriuretic peptide, epinephrine, norephinephrine, dopamine, calcitonin, and insulin.

Blood Coagulation Factors The present invention provides for the expression of blood coagulation factors by genetically modified HUCPVC. Examples of clotting factors that can be expressed in HUCPVC include, but are not limited to, Factor VII, Factor VIII, and Factor IX, and fibrinogen.

Enzymes Examples of enzymes that can be expressed in HUCPVC include butyrylcholinesterase (BChE), adenosine deaminase, glucocerebrosidase, α-1 antitrypsin, “suicide” polypeptides (eg, viral thymidine kinase (TK) ), Eg, herpes simplex virus, cytomegalovirus (CMV) or varicella-zoster virus-derived TK), hypoxanthine phosphoribosyltransferase, antioxidants (eg, manganese superoxide dismutase (Mn-SOD), catalase, copper -Zinc-superoxide dismutase (CuZn-SOD), extracellular superoxide dismutase (EC-SOD), and glutathione reductase), phenylalanine hydroxylase, nitric oxide synthase, There are fine para oxy kinase, but not limited to these.

Examples of immune regulators that can be expressed in HUCPVC include CTLA-4, VCP, PLIF, LSF-1, Nip, CD200, uromodulin, CD40L (CD154), FasL, CD27L, CD30L, 4-1BBL, CD28, CD25, B7.1, B7.2, and OX40L are not limited to these.

Receptors and ligands Examples of receptors and ligands that can be expressed in HUCPVC include T cell receptor (TCR), LDL receptor, surface-bound immunoglobulin, soluble CD4, cystic fibrosis transmembrane conductance There are but are not limited to the receptor (CFTR) and the Fc receptor.

Detectable labels Detectable labels can be expressed in HUCPVC to facilitate clinical application of these cells to mammals (eg, humans) in need thereof. A clinician can determine the amount and lifetime of labeled HUCPVC administered to a patient, for example, by isolating a blood or tissue sample and analyzing for the presence of a detectable label. Detectable labels include fluorescent molecules such as green fluorescent protein (GFP).

Metabolites and other cellular factors Examples of metabolites and other factors that can be expressed in HUCPVC include cytochrome b, cholesterol transport or metabolic polypeptides (eg, ApoE, ApoC, ApoAI), drug resistance Or antiviral resistant polypeptide (eg MDR, ribozyme, antisense RNA, antiviral protease), antitoxin agent, angiogenic peptide, tissue plasminogen activator (tPA), urine plasminogen activator (eg urokinase), hirudin , Vasoactive peptides, globin (eg, β-globin, α-globin, HbA), proto-oncogene (eg, ras, src, and bcl), and secretory peptides (eg, angiotensin converting enzyme, vascular smooth muscle calcium channel) Or add There are can) act as competitive inhibitors of Nalin receptor, but is not limited to these.

RNA Interference HUCPVC can be genetically modified to express one or more RNA interference (RNAi) molecules when administered to a patient (eg, a human). RNAi is a mechanism that inhibits gene expression by causing degradation of specific RNA molecules or preventing transcription of specific genes. The key to the RNAi mechanism is a small inhibitory RNA strand (siRNA) having a nucleotide sequence complementary to the target messenger RNA (mRNA) molecule. siRNAs are small single-stranded nucleic acid molecules that can inhibit or down-regulate gene expression in a sequence-specific manner. For example, Zamore et al., Cell 101: 25 33 (2000); Bass, Nature 411: 428-429 (2001); Elbashir et al., Nature 411: 494-498 (2001); and Kreutzer et al., International PCT Publication No. WO 00/44895; Zernika-Goetz et al., International PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No. WO 00/01846; Melo and Fire, International PCT Publication No. WO 01/29058; See Deschamps-Depallette, International PCT Publication No. WO 99/07409; and Li et al., International PCT Publication No. WO 00/44914. Methods for preparing siRNA molecules for use in gene silencing are described in US Pat. No. 7,078,196, incorporated herein by reference.

  Application of RNAi technology (eg, siRNA molecules) in the present invention can be performed in several ways, each resulting in functional silencing of the gene product in the HUCPVC population. Functional silencing of one or more endogenous HUCPVC gene products may increase HUCPVVC lifespan in vivo (eg, by silencing one or more pro-apoptotic gene products), or It may increase the expression of a therapeutic polypeptide (eg, antibody, cytokine, or hormone).

  Functional gene silencing by RNAi agents (eg, siRNA molecules) does not necessarily involve complete inhibition of the target gene product. In some instances, a slight decrease in gene product expression caused by an RNAi agent can turn into a significant function or phenotypic change in a host cell, tissue, organ, or animal. Thus, gene silencing is understood to be functionally equivalent, and the extent of gene product degradation to obtain silencing can vary between gene targets or host cell systems. Gene silencing can reduce gene product expression by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. Gene product expression is preferentially reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (ie, complete inhibition).

Genetic modification of HUCPVC Recombinant expression of non-endogenous polypeptides or oligonucleotides in HUCPVC can be performed by using several different standard gene transfer modalities. These modalities, their advantages and limitations are discussed further below. Exemplary methods for genetically modifying HUCPVC are also discussed in International Patent Application Publication No. WO 2007/128115, which is incorporated herein by reference.

Transduction (viral vector)
Transduction is the infection of a target cell (eg, HUCPVC) with a virus that allows genetic modification of the target cell. Many viruses bind to and infect mammalian cells and introduce their genetic material into host cells as part of their replication cycle. Some types of viruses (eg, retroviruses) integrate their viral genome into the host genome. This incorporates the viral gene between the host cell genes for the lifetime of the cell. For viruses modified for gene transfer, a donor gene (eg, a humanized monoclonal antibody) is inserted into the viral genome. Additional modifications are made to the virus to improve infectivity or tropism (eg, pseudotyping), reduce or eliminate the ability to replicate, and reduce immunogenicity. Newly introduced mammalian genes are expressed in infected host cells or organisms and may improve the condition or disease caused by the defective gene when the defective host gene is exchanged. Adenoviruses and retroviruses (including lentiviruses) are particularly attractive modalities for gene therapy applications because of their ability to genetically modify and take advantage of the life cycle of these viruses, as discussed below.

Adenovirus Recombinant adenovirus vectors provide several significant advantages for the expression of polypeptides (eg, antibodies, cytokines, or clotting factors) or oligonucleotides (eg, siRNA) in HUCPVC. Viruses can be prepared at very high titers and can infect non-replicating cells, resulting in high efficacy and high levels of transduction of target cells in vivo after targeted injection or perfusion . In addition, since adenoviruses do not integrate their DNA into the host genome, this gene therapy modality has a low risk of inducing spontaneous growth disorders. In animal models, adenoviral gene transfer is generally known to mediate high levels of expression over about a week. The duration of transgene expression can be extended and ectopic expression can be reduced by using a tissue specific promoter. Other improvements in the molecular engineering of the adenoviral vector itself have resulted in more sustained transgene expression and less inflammation. This includes so-called “second generation” vectors with specific mutations in additional early adenoviral genes, and the cre-lox strategy (Engelhardt et al., Proc. Natl. Acad. Sci. USA 91: 6196-6200 (1994) and Kochanek et al., Proc. Natl. Acad. Sci. USA 93: 5731-5736 (1996)), found in “gutless” vectors in which virtually all viral genes have been deleted. In addition, recombinant adeno-associated virus (rAAV) from non-pathogenic parvovirus can be used to express a polypeptide or oligonucleotide. This is because these vectors induce little cellular immune response, resulting in transgene expression that lasts for months in most systems. The incorporation of tissue specific promoters is again beneficial.

Retroviral Other viral vectors useful for delivering a polypeptide or oligonucleotide to a subject or cell are retroviruses, including lentiviruses. In contrast to adenovirus, the genetic material in retroviruses is in the form of RNA molecules, while the genetic material in their host is in the form of DNA. When a retrovirus infects a host cell, it introduces its RNA and some enzymes into the cell. This RNA molecule from retroviruses produces a double-stranded DNA copy (provirus) from the RNA molecule by a process called reverse transcription. After transport to the cell nucleus, proviral DNA is integrated into the host chromosome, permanently modifying the genome of the infected cell and any progeny cells that may arise. The ability to permanently introduce a gene encoding a polypeptide or oligonucleotide into a cell, such as HUCPVC, is a characteristic defining the retrovirus used for gene therapy. Retroviruses include a family of viruses, including human immunodeficiency virus (HIV), which includes several auxiliary proteins to facilitate lentivirus, viral infection and proviral integration.

  One problem with the use of retroviruses for gene therapy is that the integrase enzyme can insert the viral genetic material anywhere in the host genome. If genetic material is accidentally inserted in the middle of one of the host cell's prototypical genes, this gene can be disrupted (eg, insertion mutation). If the gene is a gene that controls cell division by chance, uncontrollable cell division (eg, cancer) can occur. This problem has begun to be addressed in recent years by utilizing zinc finger nucleases or by encapsulating specific sequences such as β-globin locus control regions to induce integration sites into specific chromosomal sites. Despite this consideration, retroviruses and lentiviruses have considerable utility for gene therapy applications. Current “third generation” lentiviral vectors are characterized by complete replication deficiencies, broad tropism, and increased gene transfer capabilities for mammalian cells (Mangeat, B. and Trono, D., “Lentis”). Endogenous immunity of viral vectors and antiretroviruses ", Human Gene Therapy 16 (8): 913-920 (2005) and wisnerovicz, M. and Trod, M. and Trod. And the use of HIV for biotechnology (Trnessing HIV for therapy, basic research and biotechnology), Tr ends Biotechnol. 23 (1): 42-7 (2005)). For example, LUC viruses pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) or feline endogenous virus RD114 envelope glycoprotein can be used to transduce HUCPVC (eg, Zhang et al., “Modified RD114 Transduction of bone-marrow-derived mesenchymal stem cells by using the lentivirus pseudotyped with envelope glycoproteins. .78 (3): 1219-1 29 (2004)). US Pat. No. 5,919,458, US Pat. No. 5,994,136, and US Pat. No. 7,198,950, incorporated herein by reference, are lentiviruses for genetically modifying target cells. The production and use of is described.

Other viral vectors In addition to adenoviruses and retroviral vectors, other viral vectors and techniques that can be used to introduce DNA vectors (eg, plasmids) that encode polypeptides or oligonucleotides desired for the subject or cell. Are known in the art. These include, for example, those described by Wattanapitayakul and Bauer (Biomed. Pharmacother 54: 487-504 (2000), and references therein.

Transfection Naked DNA and oligonucleotides Using naked polypeptides or oligonucleotides (eg, DNA vectors such as plasmids) that encode polypeptides (eg, antibodies, cytokines, or hormones) or RNA interference molecules (eg, siRNA or shRNA). Thus, HUCPVC can be genetically modified. This is the simplest method of non-viral transfection. There have been some successes in clinical trials performed using intramuscular injection of naked DNA plasmids, however, expression is low compared to other methods of transfection. There are other effective methods for delivering naked DNA, such as electroporation and the use of "gene guns" that use high pressure gas to shoot DNA coated gold particles to cells.

To improve the delivery of DNA vectors (eg, plasmids) into lipoplexes and polyplexes HUCPVC, the DNA can be protected from damage and its entry into the cell can be facilitated. Lipoplexes and polyplexes have the ability to protect the introduced DNA from unwanted degradation during the transfection process. Plasmid DNA can be covered with lipids on tissue structures such as micelles or liposomes. When the tissue structure is complexed with DNA, it is called a lipoplex. There are three types of anionic (negatively charged), neutral, or cationic (positively charged) lipids. Lipoplexes utilizing cationic lipids have proven useful for gene transfer. Cationic lipids naturally complex with negatively charged DNA due to their positive charge. Furthermore, as a result of their charge, they interact with the cell membrane, lipoplex endocytosis occurs, and DNA is released into the cytoplasm. Cationic lipids also protect against DNA degradation by cells.

  A complex of a polymer and DNA is called a polyplex. Most polyplexes consist of cationic polymers and their production is controlled by ionic interactions. One major difference between the methods of action of polyplexes and lipoplexes is that polyplexes cannot release their DNA packing into the cytoplasm, so that inactivated viruses are always generated for this purpose. It is to be cotransfected with an endosome lysing agent (to dissolve the endosome produced during endocytosis). However, this is not always the case. Polymers such as polyethyleneimine, like chitosan and trimethylchitosan, have their own methods for disrupting endosomes.

Hybrid methods For each method of gene transfer with disadvantages, there are several hybrid methods that have been developed combining two or more techniques. For example, virosomes combine liposomes and inactivated viruses. This approach has been shown to result in more efficient gene transfer in respiratory epithelial cells than either the virus alone or liposome alone methods. Other methods include mixing other viral vectors and cationic lipids or hybridizing the virus. Each of these methods can be used to facilitate the introduction of a DNA vector (eg, a plasmid) into HUCPVC.

Dendrimers Dendrimers can also be used to genetically modify HUCPVC. Dendrimers are highly branched macromolecules with a spherical shape. The surface of the particles can be functionalized in many ways, and many of the properties of the resulting construct are determined by the surface. In particular, cationic dendrimers (ie, dendrimers having a positive surface charge) can be constructed. In the presence of genetic material such as a DNA plasmid, charge complementation results in the temporary binding of nucleic acid and cationic dendrimer. Upon reaching that destination, the dendrimer nucleic acid complex is then incorporated into HUCPVC by endocytosis.

Biological and Chemical Agents The methods of the present invention can be used to subject exposed to or at high risk of exposure to biological or chemical agents (eg, humans such as military personnel). This results in the administration of genetically modified HUCPVC.

Bacteria The methods of the invention can be used to treat or prevent a bacterial infection in a subject (eg, a human). Bacteria causing human diseases or conditions include, but are not limited to, Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli, Klebsiella pneumoniae, Brucella, nasal bacillus, plague, and anthrax.

Viruses The methods of the invention can be used to treat or prevent a viral infection in a subject (eg, a human). Viruses that cause human diseases or conditions include flaviviruses (eg, Gadogetzgary virus, Kadam virus, Kasanur forest disease virus, Langat virus, Omsk hemorrhagic fever virus, Poissant virus, Royal farm virus, tick-borne virus Encephalitis virus, Jumping disease virus, Meevan virus, Saumarezu leaf virus, Chureny virus, Aroa virus, Dengue virus, Kedago virus, Caspa core virus, Koutango virus, Japanese encephalitis virus, Murray Valley encephalitis virus, St. Louis Encephalitis virus, Ustu virus, West Nile virus, Yaonde virus, Cocobella virus, Bugaza virus, Ileus virus, Israel Turkey meningoencephalomyelitis virus, Untaya virus, Tenba Virus, Zika virus, Banji virus, Bowbowie virus, Edgehill virus, Jugura virus, Savoia virus, Sepik virus, Uganda S virus, Weselsbron virus, Yellow fever virus, Entebe bat virus, Yokose virus, Apoi virus, Cow (Bonridge virus, ftiapa virus, modok virus, salvia virus, samperita virus, bucara bat virus, curley island virus, dakar bat virus, montanamyotis leukoencephalitis virus, phnom penh bat virus, and rio bravo virus) Togaviruses (eg, Venezuelan equine encephalitis virus (VEE), Eastern equine encephalitis virus (EEE), and Western equine encephalitis virus (WEE)), filovirus ( For example, Ebola virus (eg, Côte d'Ivoire, Reston, Sudan, Uganda, and Zaire strains), Marburg virus (eg, Angola, Ci67, Musok, Pop, and Ravnina strains), poxyvirus (eg, smallpox virus, vaccinia virus) ), Arena viruses (eg, Lassa virus (eg, Josiah, LP, and GA391 strains), IP virus, lymphocytic choriomeningitis virus, mobara virus, mopeia virus, amapari virus, flexar virus, gua Naritovirus, Junin virus, latinovirus, Machupovirus, Oliveros virus, Paranavirus, Pickindevirus, Pilital virus, Sabia virus, Tacaribe virus, Tamiamivirus, and White Otter Arroyovirus), Bunya virus (eg, Shinnonbre virus, Hantern virus, Rift Valley fever virus, Crimea Congo hemorrhagic fever virus, and Nairobi virus (eg, Dougbe virus)), herpes virus (eg, herpes simplex virus) (HSV), cytomegalovirus (CMV), Epstein Barr virus (EBV), and Kaposi's sarcoma-associated herpes virus (KSHV)), orthomyxovirus (eg, influenza virus A (eg, H5N1 avian influenza virus), B, and Influenza viruses such as C), coronaviruses (eg, severe acute respiratory syndrome (SARS) virus), and rhabdoviruses (eg, rabies virus and vesicular stomatitis virus (VSV)). But, not limited to these.

Fungi The methods of the present invention can be used to treat or prevent fungal infections in a subject (eg, a human). Fungi that cause human disease or pathology include Aspergillus, Blast myces dermatitis, Candida, Coccidioides imimitis, Cryptococcus neoformans, Histoplasma capsulatatum varieties Capsulatsum, Paracoccidioides braziliansis, Sporotrix chenkii, And zygomycete species (for example, but not limited to Absidia cholinebifera, Rhizomucorpus shirasu, and Rhizopus alhysus).

Parasites The methods of the invention can be used to treat or prevent parasitic infections in a subject (eg, a human). Parasites that cause human diseases or conditions include Toxoplasma gondii, Plasmodium falciparum (P. falciparum, Oval malaria, and P. falciparum), trypanosoma species, and Legionella species. It is not limited to only.

Chemical agent (toxin)
The methods of the invention are used to treat or prevent diseases, symptoms or conditions caused by exposure of a mammal (eg, human) to a chemical agent (eg, a toxin, chemical weapon, or blister). can do. Chemical agents that cause mammalian diseases or conditions include ricin, diphtheria toxin, E. coli enterotoxin, cholera enterotoxin, staphylococcal enterotoxin, streptococcal enterotoxin, botulinum toxin, saxitoxin, nitrogen mustard (e.g. Bis (2-chloroethyl) ethylamine), leucite (eg 2-chlorovinyldichloroarsine), sulfur mustard (eg 2-chloroethyl chloromethyl sulfide and bis (2-chloroethyl) sulfide), sarin (GB; O- Isopropylmethylphosphonofluoridate), cyclosaline (GF), soman (GD; O-pinacolylmethylphosphonofluoridate), tabun (GA; O-ethyl, n, n-dimethylphosphoramidocyanidate), VX ( O-ethyl S-2-diisopropylaminoethyl Methylphosphonothiolate), amiton, PFIB, 3-quinuclidinyl benzylate, phosgene, diphosgene, cyanogen chloride, hydrogen cyanide, chloropicrin, and acetylcholinesterase inhibitors (eg, novicoc) Not limited.

Subjects that can be treated with the genetically modified HUCPVC of the invention According to the methods of the invention for treating, inhibiting, reducing or preventing an attack with a biological or chemical agent such as a pathogen or toxin Subjects who can benefit from administration of genetically modified HUCPVC. These subjects include, for example, birds (eg, poultry such as chickens, turkeys, geese, ducks, grouse, swans, peacocks, houseflies, pigeons, and pheasants), reptiles (eg, snakes and lizards), amphibians (eg, Frogs and salamanders), mammals (eg, humans, non-human primates (eg, monkeys, chimpanzees, apes), ungulates (eg, horses, cows, goats, pigs, sheep, donkeys and deer), dogs, And vertebrates such as cats.

Dosing and Administration The present invention provides genetically modified HUCPVCs that express a therapeutically effective amount of one or more polypeptides (eg, antibodies, cytokines, or hormones) or oligonucleotides (eg, siRNA). Genetically modified HUCPVCs are intended for topical administration by parenteral (eg, intramuscular, subcutaneous, and intravenous), intranasal, topical, oral, or transdermal means for prophylactic or therapeutic treatment . In general, genetically modified HUCPVC is administered parenterally (eg, by intravenous, intramuscular, or subcutaneous injection) or intraarterial injection into the affected area. Additional routes of administration include intravascular, intraarterial, intraperitoneal, intraventricular, intradural, and nasal, ocular, intrascleral, intraorbital, rectal, topical, or aerosol inhalation administration.

  The genetically modified HUCPVC can be administered for prophylactic or therapeutic treatment. For prophylactic use, genetically modified HUCPVC is administered to a subject (eg, a human) having a clinically determined predisposition or increased susceptibility to pathogen or chemical agent (eg, chemical agent) attack To do. For example, a genetically modified HUCPVC that expresses butyrylcholinesterase (BChE) may be administered to a subject (eg, a soldier) that is recessive homozygous for the BChE gene to treat or prevent attack by a chemical agent. it can. This is because BChE polypeptides expressed in genetically modified HUCPVCs can metabolize several chemical agents.

The genetically modified HUCPVC may be administered to a subject (eg, a human) in an amount sufficient to delay, reduce, or preferably prevent the onset of a clinical disease caused by an attack by a microbial pathogen or chemical agent. it can. In therapeutic applications, genetically modified HUCPVC is already suffering from infection due to exposure to microbial pathogens or chemical agents in an amount sufficient to cure or at least partially suppress the symptoms of these agents. Administer to a subject (eg, soldier). The number of HUCPVCs suitable to serve this purpose is defined as a “therapeutically effective dose”. Effective amounts for this use may depend on the severity of the disease or condition, as well as the weight and general condition of the patient. The total number of genetically modified HUCPVCs administered to a subject in single or multiple doses according to the method of the present invention is, for example, 10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , Effective doses are probably in the range of 10 ³ to 10 ⁷ cells per dose, although there may be 10 ⁹ or more cells. The genetically modified HUCPVC is preferably administered to a subject in need thereof in a single dose. The genetically modified HUCPVC can also be applied as an initial dose, followed by one or more additional doses at 1 hour, 1 day, 1 week, 1 month, or 2 month intervals. The total effective dose of genetically modified HUCPVC administered to a subject as a single dose, either as a bolus or by infusion over a relatively short period of time, allows multiple doses to be administered over a longer period of time (eg, 4-6, 8-12, 14-16, or every 18-24 hours, or every 2-4 days, every 1-2 weeks, once a month, or once every two months) be able to. Alternatively, continuous intravenous infusion sufficient to maintain a therapeutically effective concentration in the blood is contemplated.

  A therapeutically effective amount of genetically modified HUCPVC for administration to a subject (eg, a human) according to the methods of the present invention can be determined by one skilled in the art. Factors that can be considered include, for example, the subject's age, weight, condition (eg, type of agent to which the subject is or may be exposed, biological or chemical agent to the subject) There are individual differences in the level of exposure to biological or chemical agents (for example, the severity of the effects of pathogen infections or toxins). Exposure to biological or chemical agents can be determined using well-characterized physiological markers (eg, Black and Nort, Chemical, which is incorporated herein in its entirety by reference). Warfare Agents, 2nd edition (2nd edition), John Wiley & Sons, Ltd., “Biological Markers of Exposure to Chemical Warfare Agents”, 2007. See “Biological Markers of Exposure to Chemical Warfare Agents”.

  The present invention provides for co-administration of a second genetically modified HUCPVC population to a subject (eg, a human), wherein the second HUCPVC population is one or more different poly for use in prophylactic or therapeutic applications. Expresses peptides or oligonucleotides. Alternatively, one or more mesenchymal stem cells (MSCs) that are not HUCPVCs can be administered. In this case, it is possible to genetically modify the MSC to express the polypeptide or oligonucleotide. However, it is not necessary to administer both HUCPVC or MSC populations simultaneously or in the same manner. In some cases, administration of the second population can begin immediately after the end of the administration period of the first population, or vice versa. Such a time gap between the two dosing periods can vary from one day to one week to one month or more. In some cases, two genetically modified HUCPVC populations can be co-administered first, followed by individual administration at a later time period (eg, protective against a particular virus serotype or strain). Administration of two or more HUCPVC populations individually expressing a single monoclonal antibody). In addition, the HUCPVC population can be modified to express more than one polypeptide or oligonucleotide for prophylactic or therapeutic use, thus eliminating the need for multiple doses.

  Single or multiple (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more) administration of a composition of the invention comprising an effective amount is treated It can be performed at a dosage level and pattern selected by a clinician (eg, a physician or veterinarian). Dosage and administration schedules can be determined and adjusted based on the severity or likelihood of exposure to infectious microorganisms or chemical agents. Further, subjects (eg, mammals, humans (eg, soldiers), etc.) that have been administered genetically modified HUCPVC are monitored throughout the course of treatment by methods commonly practiced by clinicians or as described herein. can do.

  One or more physiologically acceptable excipients or carriers can also be included in the composition for suitable formulations. Formulations suitable for use in the present invention can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th edition, 1985. For a brief review of methods for drug delivery, see Langer, Science 249: 1527-1533, 1990.

Other Treatment Regimes The present invention provides for co-administration of one or more therapeutic agents (eg, antibacterial agents such as antiviral compounds) in combination with genetically modified HUCPVCs. For example, additional therapeutic agents can be administered with the genetically modified HUCPVCs described herein at concentrations known to be effective for such therapeutic agents. Particularly useful therapeutic agents include, for example, cytokines, antiviral agents, immunostimulants, immunosuppressants, and vaccination vaccines.

  In some cases, the genetically modified HUCPVC and the additional therapeutic agent are at least 1 hour, 2 hours, 4 hours, 6 hours, 10 hours, 12 hours, 18 hours, 24 hours, 3 days, 7 days, 14 days Or at monthly intervals. The dosage of each component and the frequency of administration can be adjusted independently. The additional therapeutic agents described herein can be mixed with additional active or inactive ingredients, for example, in a conventional pharmaceutically acceptable carrier. The pharmaceutical carrier may be any compatible, non-toxic substance suitable for administration of the composition of the invention to the subject. Pharmaceutically acceptable carriers include, for example, water, saline, buffer and, for example, Merck Index, Merck & Co. There are other compounds described in Rahway, New Jersey. Sustained release formulations or sustained release devices can also be used for continuous administration. Additional treatment regimens can include other therapies, including changes in the lifestyle of the subject being treated.

Cytokines Cytokines can be used either as additional therapeutic agents or in combination with genetically modified HUCPVCs. Exemplary cytokines are IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, G-CSF, IL-15, GM-CSF, OSM, LIF, IFN-γ, IFN-α, IFN-β, TNF-α, TNF-β, LT-β, IL-8, MCP- 1, MIP-α, MIP-β, RANTES, TGF-β, IL-1α, IL-1β, IL-1RA, and MIF.

Antiviral Agents Antiviral agents can be used as additional therapeutic agents, either in combination with genetically modified HUCPVC or individually administered. Exemplary antiviral agents are abacavir, acyclovir, acyclovir, adefovir, amantadine, amprenavir, arbidol, atazanavir, atripura, brivudine, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirendine, emtricitabine, emtricitabine , Entecavir, Invasion inhibitor, Famciclovir, Multidrug, Fomivirsen, Fosamprenavir, Foscarnet, Phosphonet, Fusion inhibitor, Ganciclovir, Gardacil, Ivacitabine, Immunovir, Idoxlidine, Imicuimod, Indinavir, Inosine , Integrase inhibitor, interferon type III, interferon type II, interferon type I, interferon, lami Gin, lopinavir, lobilide, MK-0518, maraviroc, moroxidin, nelfinavir, nevirapine, nexabar, nucleoside analogues, oseltamivir, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitor, reverse transcriptase inhibitor, ribavirin, rimantadine , Ritonavir, saquinavir, stavudine, synergist, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, tridivir, tromantadine, trubada, valacyclovir, valganciclovir, bicrivirok, vidarabine, viramidine, zalcitabine, zanavir, and zidovir is there. Exemplary antiviral agents are listed, for example, in US Pat. No. 6,093,550 and US Pat. No. 6,894,033, which are incorporated herein by reference.

Immunostimulatory Agents When the composition of the present invention is co-administered with an immunostimulatory agent or adjuvant, it is possible to significantly improve the immunogenicity of the pharmaceutical composition. Exemplary immunostimulants include aluminum phosphate, aluminum hydroxide, QS21, Quil A (and derivatives and complements thereof), calcium phosphate, calcium hydroxide, zinc hydroxide, glycolipid analogs, octodecyl esters of amino acids , Muramyl dipeptide, polyphosphazene, lipoprotein, ISCOM matrix, DC-Chol, DDA, cytokines, and other adjuvants and derivatives thereof. Other molecules that can stimulate or costimulate cells of the immune system include CD40L (CD154), FasL, CD27L, CD30L, 4-1BBL, CD28, CD25, B7.1, B7.2, and OX40L .

Immunosuppressive agent Suppresses the immune system of the individual being treated to prevent, for example, an immune disease associated with infection (eg, dengue hemorrhagic fever syndrome) or reduce inflammation associated with exposure to a toxin May be desirable. It is also possible to use immunosuppressive agents to reduce rejection of HUCPVC administered by the host, thereby increasing the life span of these cells in vivo. Exemplary immunosuppressive agents include abethymus, deforolimus, everolimus, gusperimus, pimecrolimus, sirolimus, tacrolimus, temisirolimus, anakinra, azathioprine, cyclosporine, leflunomide, methotrexate, mycophenolic acid, and thalidomide. TNF inhibitors (eg, infliximab, adalimumab, celtrizumab pegol), alemtuzumab, aferimomab, acerizumab, atolizumab, atrolimumab, basiliximab, berimumab, bertilimumab, cedelizimab, daclizumab , Elcilimomab, ellizumab, farrimumab, fontarizumab, gartimumab, garinexumab, galinexumab, garinexumab, garinexumab, garinexumab The Relizumab, Ozurimomab, Omalizumab, Otellizumab, Pascolizumab, Pexelizizumab, Rituximab, Rituzumab, Rituzumab, Ripizumab, Ripizumab Many monoclonal antibodies that cause immunosuppression, including mabuaritox, are also known in the art.

  The following examples are intended to illustrate the present invention. These are in no way meant to limit the invention.

(Example 1)
HUCPVC expressing a humanized monoclonal antibody against the Venezuelan equine encephalitis virus (VEEV) E2 antigen.
History of VEEV as a biological weapon agent All three equine encephalitis fever viruses, Venezuelan equine encephalitis virus (VEE), western equine encephalitis virus (WEE), and eastern equine encephalitis virus (EEE) are potential organisms. It is a scientific weapon agent. Venezuelan equine encephalitis virus (VEEV) is intended to be used in weapons because of its infectivity (only 10-100 virions are needed to infect humans) and its effectiveness as a depriving agent. A particularly attractive agent. Although VEEV infection is rarely fatal, it can cause severe illness similar to influenza and therefore can be difficult to diagnose. Encephalitis fever causes long-term side effects such as brain inflammation and nervous system disorders. A possible biological attack using VEEV would be by the spray route, but since VEEV is an arbovirus, it will be most effective during periods when mosquitoes are most active. VEEV can be sprayed in a stable liquid or dry form.

  VEEV, weaponized by the United States, was an agent that deprived aggressive physical functions before the end of its biological weapons program. The Soviet Union also weaponized VEEV as an agent that deprives physical functions. Soviet scientists have also experimented with splicing the VEEV genome into smallpox virus. The result was a recombinant smallpox-VEE chimeric virus that, under the microscope, resembles smallpox virus but produces different symptoms in its host. In 1959, lyophilized vials containing VEEV were accidentally dropped by Soviet medical personnel and 20 research staff became infected. This laboratory accident and other naturally occurring outbreaks of VEEV demonstrate the infectivity of spray VEEV.

Gene Payload: Recombinant gene fusion (pRSmA116huFc) encoding humanized anti-E2 / VEEV antibody 1A4A1 human IgG ₁ heavy chain constant region and single chain fragment variable antibody was generated (Hu et al., “Venezuelan equine encephalitis virus against Humanization and mammalian expression of a murine monoclonal antibody against Venezuelan equine encephalitis virus (N). The murine monoclonal antibody 1A4A1 has been shown to recognize a conserved neutralizing epitope of the VEEV envelope glycoprotein E2 (Roehrig et al., “Neutralization on the E2 glycoprotein of Venezuelan equine encephalomyelitis (TC085) virus). The site is composed of a number of conformation-stable epitopes (The neutralizing site on the E2 glycoprotein of Venezuelan equene esophlomyleit (TC085) viro is is possibilized). . This gene fusion was placed under the transcriptional control of adeno-associated virus (AAV) to induce high levels of constitutive expression in human cells.

Evaluation of anti-E2VEEV recombinant antibody production in vitro HUCPVCs are cultured (see, eg, Ennis et al. (2008)) and transduced with pAAV-RsmA116huFc to allow expression of monoclonal antibody 1A4A1. Further experiments (eg, ELISA) confirm the amount of recombinant antibody that can be produced by genetically modified HUCPVC and describe the length of this expression. In addition, binding tests are performed to confirm the biological activity of the recombinant antibodies and their ability to neutralize VEEV virions.

In Vivo Antibody Production, Pharmacokinetics and Efficacy Stable HUCPVC transfectants shown to secrete biologically active anti-VEEV antibodies are introduced into mice by intravenous, intramuscular or subcutaneous administration To do. Use immunized normal mice. Biologically similar cells derived from bone marrow matrix are not rejected when used as xenogeneic in vivo (Plotnikov et al., “Xenograft adult human mesenchymal stem cells are a persistent biological pacemaker function in the heart of dogs. (Cenotyped in the process from the 7th to the 7th), which uses the C7 as the preparation of the 7th era of the 7th generation of the biochemically-aligned biologics of the 7 (Private data). A series of serum samples will be evaluated for anti-VEEV antibody quantity, quality, and longevity.

Pathogen Challenge Experiments After confirming therapeutic levels of anti-VEEVMAb in treated mice, mice are challenged with intranasal VEEV challenge to measure the prophylactic effect of expressed MAbs using standard methods.

(Example 2)
HUCPVC that expresses Ebola virus glycoprotein (GP) to stimulate a protective immune response.
Gene Payload: Glycoprotein of Ebola virus (Zaire strain) An rVSV vector expressing glycoprotein (GP) of Zaire Ebola virus strain Mainga (ZEBOV) is prepared (see, eg, Garbutt et al., J Virol. 78: 5458-65, 2004). And Jones et al., Nat Med 11: 786-90, 2005). Specifically, five VSV genes (nucleoprotein (N), phosphoprotein (P), matrix protein (adjacent to bacteriophage T7 promoter sequence, VSV leader sequence, hepatitis virus delta virus ribozyme sequence, and T7 terminator sequence) ( M), glycoproteins (G), and polymerases (L)) are used. Between the G and L genes, there is a unique linker site (Xho-NheI) flanked by transcription start and stop signals for the expressed Ebola virus glycoprotein. Cultured HUCPVCs are transduced with pVSV / Ebola GP vector at various multiplicity of infection.

In Vitro Glycoprotein Production and Antigenicity Evaluation Transduced HUCPVCs are tested for expression of Ebola GP using standard immunoblotting analysis, such as ELISA, Western blot, dot blot, or immunoprecipitation.

  Expression of Ebola GP when used to stimulate lymphocytes in culture using mixed lymphocyte reaction, lymphocyte proliferation assay, and cytotoxicity assay (eg, chromium release or IFN-gamma ELISPOT) Determine antigenicity.

In Vivo Glycoprotein Production, Pharmacokinetics and Efficacy By in vitro confirmation of GP expression and antigenicity, cultured HUCPVCs are administered intravenously and intraperitoneally to mice to prime anti-GP immunity. At weekly intervals after immunization, mice are sacrificed and spleen and lymph nodes (eg, fovea, groin, and upper arm) are collected. The lymphocyte proliferation and cytotoxicity assays previously described are used to determine the breadth and efficacy of adaptive anti-GP immune responses (eg, T cells and neutralizing antibodies).

(Example 3)
HUCPVC that expresses interferon and provides broad protection against many different viruses.
Recombinant expression of interferon in HUCPVC administered to patients (eg soldiers) can provide a broad range of protection against many different viruses (eg hemorrhagic fever virus). Interferon (IFN) produced by genetically modified HUCPVC can be used prophylactically or therapeutically by patients exposed to or infected with pathogenic viruses. When administered as a therapeutic agent, IFN exhibits a short in vivo half-life. Administration of genetically modified HUCPVC to express one or more IFNs (eg, IFN-α) overcomes this drawback by providing sustained release and delivery of IFN. Standard clinical administration of IFN requires frequent injections or modifications (eg, PEGylation) for rapid elimination of IFN. In this example, this problem is overcome by providing genetically modified HUCPVC to provide IFN-α.

Gene payload: Interferon α (IFN-α)
A retroviral vector (eg, lentivirus) encoding interferon-α is transduced into HUCPVC. By integrating proviral DNA into the HUCPVC chromosome, a constitutively active promoter (eg, CMV promoter) is used to induce expression and secretion of IFN-α.

In Vitro Evaluation of IFN-α Production Transduced HUCPVCs are tested for IFN-α expression using standard immunoblotting analysis, such as ELISA, Western blot, dot blot, or immunoprecipitation. The activity of IFN-α is described by Foser et al., “Improved Biological and Transcriptional Activity of Monopegylated interferon-alpha-2a isomer, Alpha-2a isomer”. 3: Confirmed by an anti-proliferation assay similar to that described by 312-319 (2003).

In Vivo Production, Pharmacokinetics and Efficacy of IFN-α Cultured HUCPVC is administered intravenously or intraperitoneally to mice by confirmation of IFN-α expression and activity in vitro. For testing of the prophylactic effect, mice are challenged for 1 week after administration of HUCPVC with virus (eg LCMV). In addition, several mice are infected with virus (eg, LCMV) for one week prior to administration of genetically modified HUCPVC to assess the therapeutic potential of these cells. Viral titers derived from serum samples are calculated during the course of the experiment and plotted against mice that did not receive HUCPVC / IFN-α treatment to determine in vivo efficacy.

Other Embodiments Although the present invention has been described with respect to specific embodiments thereof, further modifications are possible, and the present application generally follows the principles of the present invention and is known or customary within the technical field to which the present invention belongs. It is intended to encompass any variation, use, or application of the invention, including those that depart from this disclosure that are within the scope, and are adapted to the necessary features previously described herein. It will be understood that it is possible.

  All publications and patent applications mentioned in this specification are to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated herein by reference in their entirety. And incorporated herein by reference.

Claims (57)

  A method for protecting a subject against attack from a biological or chemical agent, wherein the oligonucleotides in the HUCPVC are compared to human umbilical cord perivascular cells (HUCPVC) that are not genetically modified, or Administering the HUCPVC genetically modified to increase the expression of the polypeptide, wherein the HUCPVC protects the subject from the biological or chemical agent.   The method of claim 1, wherein the subject is a vertebrate.   The method of claim 2, wherein the vertebrate is a mammal.   4. The method of claim 3, wherein the mammal is a human.   The method of claim 1, wherein the HUCPVC is allogeneic or heterogeneous to the subject.   The method of claim 1, wherein the biological agent is a pathogen.   The method of claim 6, wherein the pathogen is a bacterium, virus, fungus, or parasite.   The bacteria are Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli, Klebsiella pneumoniae, Brucella nose. ), And Bacillus anthracis.   Said viruses are Gadgets Gully virus, Kadam virus, Kadanur forest disease virus, Langat virus, Omsk hemorrhagic fever virus, Poissan virus, Royal farm virus, tick-borne encephalitis virus, Jumping disease virus, Meban virus, Saumarez Reef virus, Tyureny virus, Aroa virus, Dengue virus, Kedougo virus, Kashipa virus Cacipacore virus), Koutango virus, Japanese encephalitis virus, Mareba -Encephalitis virus, St. Louis encephalitis virus, Ustu virus, West Nile virus, Yaonde virus, Cocobella virus, Bugaza virus, Ileus virus, Israel Turkey meningoencephalomyelitis virus, Untaya virus, Tenbus virus, Zika virus, Banji virus, Boubouui virus, edge hill virus, jugura virus, savoia virus, sepic virus, uganda S virus, weselsbron virus, yellow fever virus, entebe bat virus, yokose virus, apoi virus, cowbon ridge virus ( (Cowbone Ridge virus), ftiapa virus, modock virus, salvia virus (Sal Vieja virus), Saint-Perlitau Luz, Bucasa bat virus, Kurley island virus, Dakar bat virus, Montana myos leukoencephalitis virus, Phnom Penh bat virus, Rio Bravo virus, Venezuelan equine encephalitis virus (VEE), Eastern equine encephalitis virus (EEE), Western equine encephalitis Virus (WEE), Ebola virus, Marburg virus, smallpox virus, vaccinia virus, Lassa virus, Ippy virus, lymphocytic choriomeningitis virus (LCMV), Mobara virus (Mobala virus), Mopeia Virus, Amapari virus, Flexal virus, Guanarito virus, Junin virus, Latino virus, Machupo virus, Oliver virus (Olive) ros virus), Paranavirus, Pickindevirus, Pirital virus, Sabia virus, Tacaribe virus, Tamiamivirus, and Whitewater Arroyovirus, Sinnombre virus, HanTurn virus, Rift Valley fever virus, Crimea Congo hemorrhagic fever virus, Dugbe virus, herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), Kaposi sarcoma-associated herpes virus (KSHV), influenza virus A, H5N1 bird Influenza virus, influenza virus B, influenza virus C, severe acute respiratory syndrome (SARS) virus, rabies virus, and blistering stomatitis virus It is selected from the scan (VSV), The method of claim 7.   The fungi are Aspergillus, Blastomyces dermatitis, Candida, Coccidioides immitis, Cryptococcus neoformans, Cryptococcus neoformans. var. capsulatum), Paracoccidioides brasiliensis, Sporothrix schenckii, Zygomycetes spp., Absidi 8. The method according to claim 7, wherein the method is selected from Absidia corymbifera, Rhizomucor pusillus, and Rhizopus arrhizus.   The parasites are Toxoplasma gondii, Plasmodium falciparum, Plasmodium falciparum (P. vivax), Plasmodium falciparum (P. ovale), Plasmodium falciparum (P. malaria) ), Trypanosoma spp., And Legionella spp.   The method of claim 1, wherein the chemical agent is a bacterial, viral, fungal, or parasitic toxin.   The toxin is selected from ricin, diphtheria toxin, E. coli enterotoxin, Vibrio cholerae enterotoxin, Staphylococcal enterotoxin, Streptococcal enterotoxin, and botulinum toxin; The method of claim 12.   The method of claim 1, wherein the chemical agent is synthetic or naturally derived.   The method of claim 1, wherein the chemical agent is selected from a nerve agent, a blister agent, a blood agent, and a respiratory agent.   The method of claim 1, wherein the chemical agent is selected from nitrogen mustard, sulfur mustard, acetylcholinesterase inhibitor, and lewisite.   The chemical agent is saxitoxin, bis (2-chloroethyl) ethylamine, 2-chlorovinyldichloroarsine, 2-chloroethylchloromethyl sulfide, bis (2-chloroethyl) sulfide, sarin (GB), cyclosaline (GF), soman The process according to claim 1, selected from (GD), tabun (GA), VX, amiton, PFIB, 3-quinuclidinyl benzylate, phosgene, diphosgene, cyanogen chloride, hydrogen cyanide, chloropicrin.   The polypeptide is an antibody, antibody fragment, microbial antigen, cytokine or growth factor, hormone, clotting factor, drug-resistant or antiviral-resistant polypeptide, antitoxin drug, antioxidant, receptor or ligand, immunomodulator, detectable The method of claim 1, wherein the method is selected from a label and a cellular factor.   19. The method of claim 18, wherein the cell synthesizes and secretes the antibody or antibody fragment.   19. The method of claim 18, wherein the antibody or antibody fragment is recombinant or humanized.   The method of claim 18, wherein the antibody or antibody fragment is monoclonal.   19. The method of claim 18, wherein the antibody or antibody fragment is a single chain antibody (scFv), Fab, Fab'2, scFv, SMIP, diabody, nanobody, aptamer, or domain antibody.   The cytokine or growth factor is tumor necrosis factor α (TNF-α), TNF-β, interferon-α (IFN-α), IFN-β, IFN-γ, interleukin 1 (IL-1), IL-1β, Interleukins 2-14, granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), RANTES, MIP-1α, transforming growth factor-β (TGF-β), platelet derived growth Selected from factor (PGDF), insulin-like growth factor (IGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), erythropoietin (EPO), and thrombopoietin (TPO), The method of claim 18.   The hormones are angiotensinogen, angiotensin, parathyroid hormone (PTH), basic fibroblast growth factor-2, luteinizing hormone, follicle stimulating hormone, adrenocorticotropic hormone (ACTH), vasopressin, oxytocin, somatostatin, gastrin 19. The method of claim 18, wherein the method is selected from: cholecystokinin, leptin, atrial natriuretic peptide, epinephrine, norephinephrine, dopamine, calcitonin, and insulin.   19. The method of claim 18, wherein the clotting factor is selected from Factor VII, Factor VIII, Factor IX, and fibrinogen.   The enzyme is butyrylcholinesterase (BChE), adenosine deaminase, glucocerebrosidase, α-1 antitrypsin, viral thymidine kinase, hypoxanthine phosphoribosyltransferase, manganese superoxide dismutase (Mn-SOD), catalase, copper-zinc-super 19. The method of claim 18, wherein the method is selected from oxide dismutase (CuZn-SOD), extracellular superoxide dismutase (EC-SOD), glutathione reductase, phenylalanine hydroxylase, nitric oxide synthase, and paraoxynase.   The receptor or ligand is selected from T cell receptor (TCR), LDL receptor, surface-bound immunoglobulin, soluble CD4, cystic fibrosis transmembrane conductance receptor (CFTR), and Fc receptor. 18. The method according to 18.   The immunomodulator is CTLA-4, VCP, PLIF, LSF-1, Nip, CD200, uromodulin, CD40L (CD154), FasL, CD27L, CD30L, 4-1BBL, CD28, CD25, B7.1, B7.2, 19. The method of claim 18, wherein the method is selected from OX40L.   The method of claim 18, wherein the detectable label is green fluorescent protein (GFP).   The cellular factor is selected from cytochrome b, ApoE, ApoC, ApoAI, MDR, tissue plasminogen activator (tPA), urokinase, hirudin, β-globin, α-globin, HbA, ras, src, and bcl; The method of claim 18.   The method of claim 1, wherein the oligonucleotide is selected from RNA interference (RNAi) molecules capable of inhibiting viral replication or infection.   32. The method of claim 31, wherein the RNAi molecule is a small molecule inhibitory RNA (siRNA) or a short hairpin RNA (shRNA) molecule.   The method of claim 1, wherein the subject has been exposed to the biological or chemical agent prior to the administration.   2. The method of claim 1, comprising administering to the subject a single dose composition comprising the HUCPVC.   2. The method of claim 1, wherein the HUCPVC is administered as a vaccine to protect the subject.   2. The method of claim 1, wherein the polypeptide is a cellular protein that acts as an antigen, thereby generating an immune response against the biological or chemical agent in the subject.   2. The method of claim 1, wherein the cells persist in the subject for more than a week.   38. The method of claim 37, wherein the HUCPVC persists in the subject for more than a month.   40. The method of claim 38, wherein the HUCPVC persists in the subject for more than 2 months.   The HUCPVC is intravenous, intramuscular, oral, inhalation, parenteral, intraperitoneal, intraarterial, transdermal, sublingual, nasal, buccal, liposome, fat, ocular, intraocular, subcutaneous, subarachnoid to the subject. 2. The method of claim 1, wherein the method is administered topically, topically, or locally.   The method of claim 1, wherein the HUCPVC avoids immune recognition in the subject. 2. The method of claim 1, wherein the subject is administered 10 < ¹ > to 10 < ¹³ > HUCPVC per dose. 43. The method of claim 42, wherein the subject is administered 10 < ³ > to 10 < ⁸ > HUCPVC per dose.   The method of claim 1, wherein the HUCPVC is genetically modified to express two or more oligonucleotides or polypeptides.   2. The method of claim 1, further comprising administering at least one mesenchymal stem cell (MSC) that is not HUCPVC.   46. The method of claim 45, wherein the MSC is genetically modified such that expression of an oligonucleotide or polypeptide in the MSC is increased compared to an unmodified genetically modified MSC.   43. The method of claim 42, wherein the MSC is isolated from bone marrow, umbilical cord blood, embryonic yolk sac, placenta, skin, or blood.   2. The method of claim 1, further comprising administering one or more therapeutic agents to the subject, wherein the therapeutic agent enhances or prolongs the prophylactic or therapeutic benefit of HUCPVC treatment.   49. The method of claim 48, wherein the therapeutic agent is a cytokine, antiviral agent, immunostimulatory agent, or immunosuppressive agent.   2. The method of claim 1, wherein the HUCPVC is administered with a pharmaceutically acceptable carrier or excipient.   The method of claim 1, wherein the oligonucleotide or polypeptide is endogenous to the HUCPVC.   2. The method of claim 1, wherein the oligonucleotide or polypeptide is not endogenous to the HUCPVC.   48. The method of claim 46, wherein the oligonucleotide or polypeptide is endogenous to the MSC.   48. The method of claim 46, wherein the oligonucleotide or polypeptide is not endogenous to the MSC.   A kit comprising HUCPVC genetically modified to increase the expression of oligonucleotides or polypeptides in said HUCPVC compared to non-genetically modified human umbilical cord perivascular cells (HUCPVC), wherein said HUCPVC is an organism Kit as described above, which provides protection from chemical or chemical agents.   55. Human umbilical cord perivascular cells that have been genetically modified for use in the method of any one of claims 1 to 54.   57. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the genetically modified human umbilical cord perivascular cells of claim 56, wherein the cells are the method of any one of claims 1 to 54. Wherein the pharmaceutical composition is present in a dose effective for use according to the above.